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Functional expression of an Arabidopsis p450 enzyme, p-coumarate-3-hydroxylase, in the cyanobacterium Synechocystis PCC 6803 for the biosynthesis of caffeic acid

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Abstract

Caffeic acid, which exhibits strong anticancer activities, is a natural phenolic compound found in small amounts in plants. Production of caffeic acid by bacterial systems is technically challenging due to difficulties in functionally expressing p-coumarate-3-hydroxylase (C3H), a cytochrome P450 enzyme that converts p-coumaric acid into caffeic acid. Here, we report for the first time that the cyanobacterium Synechocystis PCC 6803 is able to produce caffeic acid from p-coumaric acid upon heterologous expression of C3H. The Arabidopsis thaliana ref8 gene, which encodes a C3H, was synthesized and codon-optimized for enhanced expression in Synechocystis. Expression of the synthetic ref8 gene was driven by a native psbA2 promoter and confirmed at the transcriptional and translational levels. This heterologous pathway enabled Synechocystis to produce caffeic acid at a concentration of ∼7.2 mg L−1 from p-coumaric acid under oxygenic photosynthetic growth conditions. This study demonstrates that cyanobacteria are well suited for the bioproduction of plant secondary metabolites that are difficult to produce in other bacterial systems.

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Acknowledgments

This work was supported by the National Science Foundation (grant no. MCB1120153), Shandong Province “Taishan Scholar” Foundation (no. tshw20091014), and by the Arkansas P3 Center (pilot seed grant P3-203). This paper is dedicated to the memory of Ms. Jing Zhang, a former master's student in the laboratory, who contributed significantly to the project. The authors thank Dr. Michael Sullivan for the anti-C3H antibody (which was obtained through a Material Transfer Agreement).

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Authors and Affiliations

  1. Department of Applied Science, University of Arkansas at Little Rock, 2801 South University Ave, Little Rock, AR, 72204, USA

    Yong Xue, Stephen Grace & Qingfang He

  2. High Tech Research Center, Shandong Academy of Agricultural Sciences, 202 Gongye North Road, Jinan, Shandong Province, 250100, China

    Yan Zhang & Qingfang He

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  1. Yong Xue
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  2. Yan Zhang
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  3. Stephen Grace
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  4. Qingfang He
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Corresponding author

Correspondence to Qingfang He.

Additional information

Y. Xue and Y. Zhang contributed equally to this work.

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Fig. S1

ref8 and sref8 nucleotide and amino acid sequences employed in this work. a: ref8 is the native Arabidopsis thaliana (ecotype landsberg erecta) cDNA sequence. b: sref8 is the Synechocystis codon-optimized version of the Arabidopsis thaliana cDNA sequence. Translated amino acid sequences of ref8 and sref8 are also listed. (DOCX 14.9 kb)

Fig. S2

Codon usage optimization for ref8. The nucleotide sequence of ref8 was optimized according to the codon preferences of Synechocystis. Codons with a usage frequency of below 23 % were not used. The 23 % cutoff is shown as a horizontal line. ref8 represents the original gene (left panel). sref8 represents the synthesized gene after codon optimization (right panel) (JPEG 25 kb)

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Xue, Y., Zhang, Y., Grace, S. et al. Functional expression of an Arabidopsis p450 enzyme, p-coumarate-3-hydroxylase, in the cyanobacterium Synechocystis PCC 6803 for the biosynthesis of caffeic acid. J Appl Phycol 26, 219–226 (2014). https://doi.org/10.1007/s10811-013-0113-5

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  • DOI: https://doi.org/10.1007/s10811-013-0113-5

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